Novel bead-based platform for direct detection of unlabelled nucleic acids through Single Nucleobase Labelling
Graphical abstract
Introduction
MicroRNAs (miRNAs) are small non-coding RNAs of 19–24 nucleotides in length that regulate gene expression by base pairing with the 3′-untranslated region of a target gene's messenger RNA (mRNA), leading to degradation and/or translational repression of that gene [1], [2], [3]. miRNAs are implicated in many biological events and their deregulation is associated with many serious disease states [4], [5].
miRNAs while present in biological fluids in a stable and reproducible manner are differentially expressed under pathological conditions, such that the expression patterns of circulating miRNAs can thus be used as fingerprints for various diseases [6], [7]. As such miRNAs possess ideal characteristics of a biomarker – i.e., they can be specific to the disease or pathology of interest, reliably indicate disease before clinical symptoms appear, be sensitive to changes in the pathology (disease progression or therapeutic response), and also allow detection from samples of biological fluids (e.g. blood, urine) [8], [9], [10], [11], [12], [13], [14]. Additionally, circulating miRNAs are remarkably stable in body fluids, resisting ribonucleases and variations in physicochemical conditions such as pH [15].
Recently, microRNA122 (miRNA122) has been found to be substantially elevated in the plasma of patients with drug-induced liver injury (DILI), and can be detected much earlier than current clinical biomarkers such as alanine aminotransferase (ALT) and aspartate aminotransferase (AST) [16], [17]. ALT and AST levels increase 12–16 h post overdose of drugs like acetaminophen, while miRNA122 can detect liver injury within 4 h [18]. Moreover, miRNA122 has been demonstrated as an in vitro marker of drug-induced cellular toxicity for acetaminophen and diclofenac, with sensitivity similar to conventional assays that measure lactate dehydrogenase activity and intracellular adenosine triphosphate. Thus, miRNA122, as a biomarker, also has the potential to be used during early phases of the drug development processes [19].
Therefore, the development of a rapid test for miRNA122 would be useful in healthcare and drug development [18], [20], [21]. However, technical difficulties to perform robust and comparable profiling of circulating miRNAs have impeded progress to develop an approved clinical diagnostic assay [22], [23]. A reliable detection platform which removes laborious sample-preparation steps (e.g., enzymatic steps for PCR-based amplifications) would represent a step forward, and allow miRNAs to be part of the approved clinical diagnostic test arsenal.
To date, most of the detection methods developed for direct detection of unlabelled nucleic acids are based just on hybridisation events using tagged probes. There are sandwich hybridisation approaches with two probes [24], which can also be assisted by ligases to elongate short sequences of miRNAs [25], [26], methods based on triple-stem DNA probes [27] and methods which use a single probe to create specific duplexes which can then be identified either by modified surfaces, [28], [29] or p19 protein that specifically recognises nucleic acid duplexes [30], [31]. These molecular assays based on hybridisation without further molecular recognition are then integrated within different detection systems such as electrochemical sensors [27], [29], [31], [32], [33] and fluorescence-based platforms [25] with variable limit of detections. In particular, there are three reports which claim the direct detection of miRNA122 using electrochemical sensors with limit of detections of 2.7 pmoles [32], 1 pmole [33] and sub-attomole [27] respectively.
This work describes the potential for direct quantification of miRNAs by combining a PCR-free approach that enables detection of nucleic acids using dynamic chemistry [34], [35], [36], combined with a bead-based platform (Luminex®) already in wide clinical use [37], [38], [39], [40]. Nucleic acid analysis by dynamic chemistry has already been successfully used for genotyping Single Nucleotide Polymorphisms (SNPs) [35] and here we describe the application of this technology to detect circulating miRNAs. Its high specificity together with its advantage of PCR- and label-free testing of nucleic acids is applied here to detect unlabelled miRNA sequences through what we describe as ‘Single Nucleobase Labelling’ (SNL) defined as “a non-enzymatic labelling of nucleic acids using dynamic chemistry” and which is ideal for short nucleic acid strands.
Nucleic acid analysis by dynamic chemistry harnesses Watson–Crick base pairing to template a dynamic reaction on a strand of an Abasic peptide nucleic acid [PNA; a DNA mimic in which the sugar-phosphate backbone is replaced with N-(2-aminoethyl) glycine]. This is achieved by hybridising an Abasic PNA probe to the target nucleic acid strand such that a nucleobase-free position on the PNA (a so-called ‘blank’ position) lies opposite to a nucleotide on the nucleic acid strand. The reversible reaction, between an aldehyde-modified nucleobase (SMART Nucleobases) and a free secondary amine on the PNA probe, generates an iminium intermediate which can be reduced to a stable tertiary amine, (reaction known as reductive amination). Four iminium species (one for each base) will be thus generated, but the one with the correct hydrogen bonding motif (obeying Watson–Crick base-pairing) will be the most thermodynamically stable product [34] (Fig. 1). Moreover, complementary nucleic acid strands act also as catalysts as accelerate the rate of the reductive amination. When there is not complementary nucleic strands, reductive aminations do not happen within the assay timeframe. In summary, this technology requires two specific molecular events to create a signal, (i) perfect hybridisation between nucleic acid strands and Abasic PNA and (ii) specific molecular recognition, through Watson–Crick base-pairing rules, by the SMART Nucleobase (Fig. 1).
With these features in mind, we aimed to use this technology for the direct detection and quantification of miRNAs. In this case, native miRNAs act as template molecules which drive the specific incorporation of a labelled SMART Nucleobase onto a specific Abasic PNA. In cases where there are other nucleic acids which are not fully complementary to the Abasic PNA, reaction does not happen. Quantification is also possible as the yield of the reaction depends on the amount of templating miRNA.
To achieve the merging of this technology with Luminex® bead-based detection platform, Abasic PNA probes complementary to target miRNAs were covalently bound to Luminex® microspheres (Fig. 2). Labelling was achieved via an aldehyde-modified nucleobase tagged with biotin (Fig. 2, Step 1) and Streptavidin-R-Phycoerythrin Conjugate (SAPE) (Fig. 2, Step 2) to allow microspheres to be read using a Luminex® MAGPIX® platform (Fig. 2, Step 3). Validation of the assays developed was further confirmed using two alternative technologies, i.e., flow cytometry and confocal microscopy.
Section snippets
General
Carboxylated magnetic microspheres (MagPlex, MC10012) and MagPlex-TAG microspheres with surface-bound oligonucleotide (MTAG-A012) were obtained from Luminex® Corporation (Netherlands) and stored at 4 °C in the dark. All chemicals were obtained from Sigma Aldrich and used as received. SCD buffer was prepared from 2×saline sodium citrate (SSC) and 0.1% sodium dodecyl sulphate (SDS) with the pH adjusted to 6.0 using HCl. All synthetic DNA oligomers (desalted) were purchased from Microsynth AG
Polyanionic Abasic PNA probes improve hybridisation efficiency
It is well known that standard PNA molecules tend to adopt collapsed conformations, due to their neutral backbone and hydrophobic interactions between nucleobases [42], [43], [44]. In the case of Abasic PNA probes bound to polystyrene microspheres, these conformations were anticipated to be further enhanced due to hydrophobic interactions between polymers and PNA oligomers. In order to resolve this, we hypothesised that adding anionic groups across the probe backbone would reduce this tendency,
Conclusions
A “Proof of Concept” study for the integration of Single Nucleobase Labelling with Luminex xMAP® technology for the detection of miRNA122 is presented. Effective conjugation of the DGL probes to MagPlex microspheres allowed detection of miRNA122 via dynamic chemistry demonstrating the potential for rapid and accurate profiling of miRNA122 in particular and miRNAs in general. A detection limit of 15 fmoles (0.3 nM) together with high specificity and the capacity to differentiate sequences
Acknowledgements
This research was supported by a Scottish Enterprise SMART Grant (United Kingdom) number SMART/13/070/13/9259. JJDM thanks Spanish "Ministerio de Economía y Competitividad, Spain" for a Ramon y Cajal Fellowship. This research was also partially supported by: a) Marie Curie Career Integration Grants within the European Community "Seventh Framework Programme, Belgium" (FP7-PEOPLE-2011-CIG-Project Number 294142) to RMSM; b) Marie Curie Career Integration Grants within the European Community "
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2019, TalantaCitation Excerpt :An alternative approach for nucleic acid testing by dynamic chemistry (also known as Chem-NAT), which harnesses Watson-Crick base pairing to template a dynamic reaction on a strand of abasic peptide nucleic acid (PNA) probes [12] and using reactive nucleobases (SMART-NBs) has been developed by our group. During the past years, we have validated this approach to detect DNA mutations [13–16] and miRNAs without the need of using PCR [17–20]. Hence, new applications that fitted into the actual chemical biological and biomedicine demands are being developed.
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2018, TalantaCitation Excerpt :We previously reported a multiplex assay for fluorescent miRNA detection using the Luminex coupled with stem–loop probes, where one specific streptavidin-phycoerythrin (SA-PE) of high molecular weight needed to be employed to alleviate background interference instead of commonly used SA-PE [18]. Venkateswaran et al. [24] developed a dynamic chemical xMAP approach for direct quantification of miRNAs with a limit of detection of 15-fmol miRNA 122. To further enhance detection method sensitivity, various miRNA analysis methods have emerged based on nucleic acid amplification with the help of enzymes, such as RT-PCR [12], rolling circle amplification (RCA) [25,26], isothermal exponential amplification reaction (EXPAR) [27], and loop-mediated isothermal amplification (LAMP) [28].
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These authors contributed equally and should be considered co-first authors.